TREM2 as a Prognostic Biomarker for Osteosarcoma Microenvironment Remodeling

The tumor microenvironment (TME) acts as a crucial role in the occurrence and development of osteosarcoma (OS). Despite this, the mechanism controlling the components of immunity and stroma in the tumor microenvironment remains a mystery. To conduct this study, we download and collate transcriptome data from the TARGET database, whose full name is Therapeutically Applicable Research to Generate Effective Treatments, as well as available clinical information of OS. The CIBERSORT and ESTIMATE methodology are used to acquire the proportions of components of immunity and stroma and tumor-infiltrating immune cells (TICs). Protein-protein interaction (PPI) networks and Cox regression analysis are used to select differentially expressed genes (DEGs). A prognostic biomarker is determined by intersecting univariate COX and PPI results, which lead to the finding of Triggering receptor expressed on myeloid cells-2 (TREM2). Based on the next analysis, TREM2 expression is positively correlated with OS survival time. Immune function-related genes have enrichment in the group with high expression of TREM2, according to gene set enrichment analysis (GSEA). The percentage of TICs by CIBERSORT methodology revealed that the expression of TREM2 is positively associated with follicular helper T cells, CD8-positive T cells, and M2 macrophages and negatively correlated with plasma cells, M0 macrophages, and naive CD4-positive T cells. All results suggest a possible integral role of TREM2 in the immune-related events of TME. Therefore, TREM2 may be a potential indicator of remodeling of TME in osteosarcoma, which is useful and helpful in predicting the clinical prognostic outcome of OS patients and provide a unique perspective for immunotherapy for OS.


Introduction
Chiefy afecting children and young adults and occupying about nine percent of cancer-related deaths in youngsters whose age range is between 10 and 24 years old [1,2], the exact cause of osteosarcoma is still unclear as a primary malignant bone tumor. In addition, the local invasiveness and metastasis of osteosarcoma remain an enormous challenge of therapy and poor prognosis [3]. With the advent of adjuvant and neoadjuvant chemotherapy, the fve-year survival rate of OS had a substantial increase which is upto about 70% since the 1970s, but after lung metastasis, the fve-year survival rate still maintains as low as 20-30% [4]. In addition to this, osteosarcoma is highly heterogeneous which makes the prediction of treatment outcomes complicated [5]. Te OS includes distinct histological subtypes: osteoblastic, chondroblastic, fbroblastic, giant-cell rich, epithelioid, small-cell, and telangiectatic types [4]. Terefore, exploring new diagnostic and predictive biomarkers and validating more therapeutic targets are continuously essential and critical.
Acting decisive roles in tumor occurrence, progression, metastasis, and sensitivity to therapy, TME has aroused tremendous interest in basic and clinical research as a therapeutic target in cancer [6]. Resident stromal cells and recruited immune cells are the primary component of TME in OS. Tere is convincing evidence to prove that the stromal cell acts a prominent role in angiogenesis and the remodeling of extracellular matrix in tumors [7]. Te occurrence, growth, and progression of tumors are critically afected by the mutual efects between host tumor cells and stromal cells. However, the stromal components of diferent tumors vary widely. Te understanding of the mechanism of crosstalk among tumors is still at a low level [7]. In the meantime, several studies keep close tabs on how immunerelated cells impact tumor occurrence, growth, and progression. An increasing number of research studies reveal that TICs acted as an up-and-coming indicator for the understanding and therapeutic efects of TME [8]. Studies have shown that osteosarcoma's immune environment is primarily composed of T-lymphocytes and macrophages. Osteosarcoma cells can control the recruitment, diferentiation, and development of immune-infltrating cells, which results in a local environment of immune tolerance. Tis kind of environment is favorable to the development of tumors, the resistance of drugs, and even metastases [9,10]. Terefore, to properly demonstrate the mechanism of TME immune and stromal components regulation, precise genetic analysis is a research hotspot as well as a challenge.
In our study, CIBERSORTand ESTIMATE methodology is used to count on the proportions and composition of the components of immunity and stroma of OS patients from the TARGET database and selected interesting biomarker TREM2. Several researchers identifed the TREM2 receptor as a dominating signaling hub of pathology-induced immunity, which can sense tissue damage and activate robust remodeling immunity as responding to it [11]. By playing a part in tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), TREM2 participated in facilitating an immune-suppressive TME in numerous cancers, including lung cancer, gastric cancer, and glioma [11][12][13]. In our study, embarking on a comparison between components of immunity and stroma in TME, diferentially expressed genes (DEGs) are generated, which revealed that TREM2 may be a potential biomarker of TME remodeling in osteosarcoma.

Data Collection and Processing
2.1. Data Source. All data of transcriptome RNA-seq of 101 OS samples and clinical data (including age and sex) of 253 clinical cases are downloaded and collated from the TAR-GET database (https://ocg.cancer.gov/programs/target) on May 24, 2022. In the genetic screening phase, we used all transcriptomic data, but in the prognostic analysis, we used only those data that had both transcriptomic data and survival status (survival status and survival time). After integration, a total of 95 samples had both transcriptome and survival data. Tere were 55 men and 40 women. Eleven patients were younger than 10 years, 62 were between 10 and 18 years, and 22 were older than 18 years.

Calculation of Tree Kinds of Score.
To estimate the components of immunity and stroma in TME for every sample, the ESTIMATE algorithm is loaded with estimate package [14] in R software (version 4.2.0). Te three kinds of scores (ImmuneScore, StromalScore, and ESTIMATEScore) increase with the increase of each of the three levels (immunity-related, stroma-related, and the summation of both), respectively. Te larger the scores are, the higher the respective composition of the corresponding TME components is.

Survival Analysis.
We combined the three kinds of scores in TME with survival information of OS patients, using the Limma package in R. On account of the median value immune score and stromal score, ninety-fve OS patients are split into two diferent groups, low-and high-score groups, respectively. Using the survival and survminer packages in R software, survival and survminer analyses are calculated. Survival curves are plotted using the Kaplan-Meier methodology, and statistical signifcance is ascertained by log-rank test; P < 0.05 is accepted as significant statistically.

Identifcation of Diferently Expression Genes between the
Low and High Groups. Te median value allows the sample to be divided into two equal parts, so we use it as the split line. One hundred and one patients are distinguished as low or high scores, respectively, in comparison with the median ImmuneScore and StromalScore values. Diferences between high-and low-scoring samples are achieved by using the R and limma package, also low and high subgroups are compared to obtain the corresponding diferentially expressed genes. Genes with FDR <0.05 and log 2 -transformed fold change >1 (high subgroup/low subgroup) are regarded as signifcantly diferentially expressed genes.

Enrichment Analyses of GO and KEGG.
Te enrichplot, clusterProfler, and ggplot2 packages of R software are used to classify 118 DEGs according to genomic annotation information, i.e., gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). Genomic annotation information with both p and q values < 0.05 are regarded as an important and statistically signifcant role in the development and progression of osteosarcoma.
2.6. Heatmaps. R with the heatmap package is applied to establish the heatmap of DEGs.

Te Diference Analysis of Scores with Clinical
Characteristics. Data on clinical information of OS patients are also of interest. R software is used to perform statistical analysis, and Wilcoxon or Kruskal-Wallis rank sum tests are used to determine whether there are statistical diferences between clinical indicators between two groups.

Analysis Process of Tis Study.
Tis study can be divided into two major steps: the discovery of TREM2 and the follow-up study of TREM2 ( Figure 1). First, osteosarcoma tissue consists of tumor cells and stromal cells, which correspond to ImmuneScore and StromalScore. Each score is used to divide samples into two groups, respectively, using the median value as the cut-of value, and the intersection of DEGs between the high and low groups of each score is used for subsequent PPI and regression analysis, while the intersection of PPI and regression analysis results in turn, eight key genes (ITGAM, HLA-DMA, LY96, C1QA, C1QB, C1QC, TREM2, and C3AR1) are identifed. TREM2 is used as our gene of interest for subsequent studies including survival analysis, GSEA, and analysis of immune-related functions.

Scores Are Associated with OS Patient Survival and Clinical
Characteristics. An important indication of whether the immune and stromal ratios are signifcant in patients with osteosarcoma is the relationship with survival, so we performed a Kaplan-Meier analysis of three kinds of scores, and not surprisingly, the scores correlated positively with survival (Figures 2(a) and 2(b)). To assess the combined composition of two components in TME, we add Immu-neScore and StromalScore to get ESTIMATEScore (Supplement Table 1). Despite the result showing there is no signifcant correlation between ESTIMATEScore and the overall survival rate (Figure 2(c)), its p value is still less than 0.1. Tese entire results implied that the composition of TME is clinically important and the compositional aspects of TME can forecast patients' prognosis of OS, especially immune and stromal components. In addition to the survival rate, it is worth discussing whether these three kinds of scores are correlated with other clinical indicators such as age and gender. Te results indicated that gender is signifcantly correlated with Immu-neScore and ESTIMATEScore in patients (P < 0.05, Figures 2(d) and 2(f )), except StromalScore (p � 0.1, Figures 2(e)), while age is not signifcantly correlated with any score (p > 0.05, Figures 2(g)-2(i)). We found that scores in female patients are higher than in male patients.

Immune-Related Genes Are Mainly Shared DEGs between
the ImmuneScore and StromalScore. Analysis of comparing patients between low and high scores is executed to ascertain if there are defnitive genetic profle alterations of components of immunity and stroma in TME. Eight hundred and ninety DEGs (Five hundred and twenty-nine downregulated and three hundred and sixty-one upregulated genes) are received by comparing two groups (low-and high-ImmuneScore patients), with the median value as the cutof (Figures 3(a), 3(c), 3(d), Supplement Table 2). Correspondingly, fve hundred and thirty-one DEGs (Two hundred and twenty-four downregulated and three hundred and seven upregulated) are received from the StromalScore Table 3). Furthermore, twenty-nine low-score downregulated genes and eighty-nine high-score upregulated genes are cross-linked between the ImmuneScore and StromalScore by an analysis of Venn diagrams (Figures 3(c) and 3(d), Supplement Table 4). Te entire DEGs (118 genes in all) are deemed as determinants of status in TME. GO enrichment analysis results give evidence that the DEGs ordinarily have a corresponding in terms linked to immunity, including innate and acquired immunity ( Figure 3(e), Supplement Figure 1A and 1B). Te KEGG enrichment analysis similarly gives evidence of that DEG enrichment in the disease spectrum is related to the immune system, including infection and autoimmune disease ( Figure 3(f ), Supplement Figure 1C and 1D). Hence, the overall function of diferentially expressed genes appears to have a corresponding immune-related event, hinting the participation of immunity-related elements is a principal signature in the TME of OS.

Relationships between TREM2 and Survival Time and Clinical Characteristics in OS Patients.
Based on previous report, we chose TREM2 for further study [15]. According to the median expression of TREM2 gene, we separated the OS patients into two groups, low-and high-expression TREM2 expression groups. Tere is a signifcant diference of survival rate statistically between two groups by the high TREM2 expression group has a higher survival rate than patients with corresponding low expression (Figure 4(e)). What's more, there is no statistical diference between TREM2 expression and clinical characteristics (Supplement Figure 2).

TREM2 as a Potential Indicator of TME Remodeling.
Taking the fact that the levels of TREM2 expression have positive correlation with OS patient survival into consideration, these two groups are in comparison in GSEA.
Hallmark and C7 sets of both demonstrated that the groups with high expression of TREM2 have observably more enrichment in immunity-related gene sets, suggesting immunity-related functions, such as the complement response, allograft rejection, IL6/JAK/STAT3 signaling, and acquired immunity are substantially more vibrant (Figures 5(a) and 5(b)). Terefore, it is implicit that the status of the TME can be mirrored by the TREM2 expression.

Correlation Analysis of the Levels of TREM2 Expression and TICs.
To move forward a single step in confrming the relevance between TME and expression levels of TREM2. CIBERSORT methodology is utilized to acquire the immune subpopulation composition of tumor-infltrating. Te establishment of twenty-two kinds of immunity-related cell profles is executed as follows ( Figure 5(c)), and the relevance among TICs is fgured up (Figure 5 T cells gamma delta

Discussion
In the current study, genes of the tumor microenvironment that related to the survival of OS patients from the TARGET database are what we attempted to appraise. TREM2 is appraised to be engaged in immunity-related activities. More signifcantly, a battery of research on bioinformatics revealed that TREM2 is a prognostic biomarker for osteosarcoma microenvironment remodeling.
Te tumor microenvironment played a pivotal part in tumorigenesis and its progression. It is of strategic meaning to detect the underlying therapeutic targets which can contribute to the remodeling and facilitating the transition of the tumor microenvironment from a developmental state to an inhibitory state.
Numerous research studies had elucidated the signifcance of tumor microenvironment in tumorigenesis [16]. In osteosarcoma tumor microenvironment-related literature, we take notice that the connection between the score of immunity and survival state has been investigated, and C3AR1, PPARG, PDK1, IGHG3, and C1Q are recognized as prognostic biomarkers [17][18][19]. Te immune components in TME sever the purpose of the prognosis of patients by analyzing the OS data in the TARGET database. In particular, the composition of immunity and stroma in TME has a strong correlation with the overall survival in OS patients. Tese results demonstrated and emphasized the importance of pursuing the connection between stromal cells and tumor cells, which will give a novel perception for discovering and developing more efcient therapy. What's more, this paper also substantiated that TICs have relevance with the clinical prognostic outcome of OS [20]. Te relevance ofers a brand new theoretical footstone for the evolution of more efcient immunotherapeutic methods.
For the last few years, tremendous progress was acquired in immunotherapy, and the inhibition of immune checkpoint inhibitors (ICIs) in OS made signifcant progress [21,22]. However, the inhibition of immune checkpoint inhibitor (ICI) immunotherapy for osteosarcoma (OS) is severely restricted by the lacking of immunogenicity and poor T cell infltration [23,24]. Terefore, the immunotherapy of OS is in urgent need of some novel candidate exploitation. Here, the decreased expression of TREM2 has a signifcant association with poor prognosis by analyzing the transcriptomic of OS in the TARGET database. Consequently, we will center on the relevance between the expression of TREM2 and TME to supply a novel treatment idea for OS immunotherapeutic methods.
As a dominating signaling hub of pathology-induced immunity, TREM2 catch the attention of the leading role of myeloid cells in various pathological processes which can mediate immunosuppression [25]. Many markers of tissue injury are ligands for the TREM2 receptor, and binding of the TREM2 receptor and ligand contributes to cell survival and resistance to infammation, afecting cell phenotype by regulating phagocytosis and metabolism [15]. In cancer research, TREM2 is observed in macrophages beyond 200 cancer cases of humans in fostering an immune-suppressive TME [15]. Tere, TREM2 is perhaps a biomarker to alter tumor bone marrow infltration and reinforce immunotherapy of ICIs [25].
CIBERSORT methodology is applied to accomplish the analysis of the proportion of TICs and completed the composition of twenty-two profles of immune cells. Te results exhibited that macrophages accounted for the highest proportion in the TME of OS, especially M2 macrophages. Te fraction of M2 macrophages in high expression groups of TREM2 is higher, which may have a relation to the immune-suppressive TME. In addition, CD8-positive T cells and follicular helper T cells have a positive correlation with the diferential expression of TREM2. Plasma cells, naive CD4-positive T cells, and M0 macrophages have a negative correlation with the expression of TREM2. All results suggest that the diferential expression of TREM2 is linked to the levels of immune cell infltration and is a critical target for ameliorating the prognosis of OS. As an attractive biomarker for modulation of individual immunotherapy who are intractable to therapy of ICIs and have a TME rich in TAM, TREM2 is tightly associated with TAMs [26].
Applying the ESTIMATE algorithm, functional enrichment analysis is applied to acquire the gene of the tumor microenvironment of OS in the TARGET database. TREM2 catches our eye as a potential prognostic biomarker for OS patients. What is of interest is that, although TREM2 may mediate the immunosuppressive tumor microenvironment through macrophage M2 polarization, its expression level has positive relevance with the overall survival time of osteosarcoma patients. Further research is indispensable to disclose the mechanism of regulating and exploit novel immunotherapeutic strategies.

Data Availability
Te datasets analyzed for this study can be found in the TARGET database (https://ocg.cancer.gov/programs/ target).

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
SZL conceived and designed this study. SZL, CZY, and JY performed data processing and analysis and wrote the manuscript. JH, ZZP, and YH performed a literature search. LB, XW, and XJR performed a review and specialty consultation. All authors read and approved the fnal manuscript. Zhi-Long Shen, Zhao-Yu Chen, and Yong Ji contributed equally to this work and share the frst authorship.  Table 1: immune infltration score. Te row is the ID of the sample and the column is the score, including StromalScore, ImmuneScore, and ESTIMATEScore. Supplementary Table 2: diferentially expressed genes between high and low ImmuneScore groups. Te row is the gene name, and the column is the fold change after taking the logarithm with the base of 2 and the adjusted P value in turn. Supplementary Table 3: diferentially expressed genes between high and low StromalScore groups. Te row is the gene name, and the column is the fold change after taking the logarithm with the base of 2 and the adjusted P value in turn. Supplementary Table 4: common diferentially expressed genes shared by ImmuneScore groups and Stro-malScore groups. Te row is the gene name and the column is the fold change after taking the logarithm with the base of 2. (Supplementary Materials)